In the late 1970's, methods were developed which allowed the introduction and expression of foreign or exogenous DNA into mammalian cells in culture. This technique, known as transfection or transformation, is a powerful method for examining the function and expression of various genes of mammals and many mammalian viruses.
By introducing exogenous DNA of interest into a cell, and monitoring the expression of a gene contained in the DNA, the gene can be analyzed in greater detail than in a cell where it is naturally expressed. For example, mutations and/or deletions of defined regions of a gene can be used to identify DNA elements within the gene that are important for the regulation of its expression. Such studies have lead to the discovery of novel transcriptional regulatory elements for genes as well as various RNA processing and translational signals. In addition, the introduction of an exogenous gene into a cell line can be used to study the effect of that gene on the growth of the cell or the effect of drugs and other agents on the expression of the transfected gene.
Transfection techniques have also been used to isolate genes by transfecting cells with a pool of genomic DNA and selecting the desired cells using genetic complementation techniques. Various mammalian genes, including the chicken thymidine kinase gene, the human hypoxanthine guanine phosphoribosyl-transferase (HPRT) gene, the human thymidylate synthase gene, the human transferrin receptor gene, a human DNA repair gene, and new oncogenes, have been isolated using this technique.
The broad applicability and usefulness of transfecting DNA into mammalian cells has led to the development of a number of protocols for performing transfections, many of which involve the use of either calcium phosphate or DEAE-dextran (or its analogs) as a carrier to promote the uptake of exogenous DNA by cultured mammalian cells. Other methods have used "lipofection" techniques, which incorporate the use of synthetic cationic lipids to effect the transfection. Osmotic shock of the cells or treatment of the cells with lysosomal inhibitors has been used in an attempt to enhance transfection efficiencies. Other attempts to increase the efficiencies have used high-voltage electric pulses to create pores in the cell membranes to increase the efficiency of DNA uptake by the cells.
The transfection efficiencies obtained by these methods are relatively low, ranging from 0.001% to 1%, depending on the cell line used as a recipient. (Transfection efficiency is often expressed as either: the % of cells which have acquired the characteristic conferred by the introduced gene, as may be determined by staining the treated cells; or a measure of the aggregate amount of DNA taken-up by the cells as determined by assaying for a gene product encoded in the transfected DNA.) These low transfection frequencies have limited the application of the technique to a few cell lines which exhibit high transfection efficiencies.
The ability to transfect a wide variety of cell lines, such as those carrying mutations of interest, would facilitate the analyses of the regulatory regions controlling the expression of genes, allow the isolation of genes by genetic complementation, or the cloning of CDNA sequences on the basis of their expression.
Recently, there have been described higher-efficiency transfaction techniques which use a modification of the calcium phosphate-mediated transfection method. The modified calcium phosphate-mediated cell-transfection technique is performed in a BES (N,N-bis(2-hydroxyethyl)-2-amino-ethane-sulfonic acid)/phosphate-buffered saline, adjusted to a pH of about 6.9 to 7.0. An equal volume of the BES/phosphate-buffered saline is added to a solution comprising DNA and 0.25 M CaCl.sub.2, mixed, and incubated for 10 to 20 minutes at room temperature. The DNA/CaPO.sub.4 precipitate which forms is then added dropwise to each 60 mm plate of cells incubated in 5 ml of media containing fetal bovine serum (FBS). The cells are initially incubated in an atmosphere of 3%, by volume, CO.sub.2 at 35.degree. C. for about 15 to 24 hours (Chen and Okayama, Molecular and Cellular Biology, 7, 2745-2752, 1987). With this transfection method, cells are grown in 10%, by volume, FBS during the transfection procedure. Under these conditions, many common cultured mammalian cell lines have reportedly been transfected with efficiencies as high as 10 to 50%. It should be noted that the results for determining transfection efficiencies reported from this method required long growth periods of the cells prior to quantification, thus allowing "daughter" cells from the originally transfected cells to "reseed" the plate, resulting in an artificially high measure of transfection efficiency.
While such techniques represent an improvement over the previously known techniques, it is desirable that a transfection method is provided which results in higher transfection efficiencies, as determined by the number of cells which take up DNA and the amount of DNA taken up by the cells. It is also desirable that the time taken to Rerform the transfection procedure is reduced so that results of the transfection can be evaluated in a shorter time period than is currently required.
In view of the foregoing, there is a need for a method of transfecting cells which results in high transfection efficiencies, with a wide variety of mammalian host cell lines, and which requires only a short period of time for cells to take up exogenous DNA.